The present invention relates to electrophysiology and in particular to “patch clamping” for investigating ionic and molecular transport through cellular membranes via ion channels and pores. Invention permits construction of microscale pores that may be readily sealed to cellular membranes and controlled in temperature with respect to the surrounding liquid bath.
Ion channel investigation using patch clamps plays an important role in drug discovery and preliminary drug screening or evaluation, for example, by providing a model that shows an effect of a drug on ion channels. Experiments performed with patch clamps can be used to test for adverse effects or search for positive therapeutic effect in the treatment of ion channel related diseases.
Drug screening can require a large number of ion channel measurements. In current practice, planar patch clamps are preferable because they allow parallelization of multiple samples on a substrate, often referred to as a wafer, chip, or well-plate, and facilitate measurement automation. Each sample includes a cell or cell wall that is positioned so that an ion channel in the cell or cell wall is aligned with a pore at the sample site. The cell or cell wall is sealed to the patch clamp substrate in a manner that allows ion channel investigations with only a small amount of electrical current, possible because of a high resistance seal between the patch clamp substrate and the cell wall (a gigaohm seal or gigaseal). Gigaohm seals achieved using on-chip patch clamp procedures usually have electrical resistance values of about one gigaohm, with resistance values of up to about 5 gigaohms being achieved in some instances.
Planar patch clamp substrates can be made from, for example, silicon (and other semiconductors), Teflon®, PDMS (polydimethylsiloxane), PSG (phosphosilicate glass), or glass. While such materials prove suitable for many planar patch clamp implementations, a single crystal quartz (quartz) material can be particularly desirable for making planar patch clamp substrates. Quartz exhibits particularly high electrical insulating properties and is piezoelectric.
Traditionally, micromachining of glass and quartz is performed using a combination of lithography and reactive ion etching (RIE). However, RIE techniques require multiple steps and are relatively slow processes. US patent application 2011/0111179 entitled: “Laser Drilling Technique for Creating Nanoscale Holes” and US patent application 2010/0129603 entitled: “Retro-Percussive Technique for Creating Nanoscale Holes”, both assigned to the assignee of the present invention and hereby incorporated by reference, teach improved methods for micro-machining small holes (e.g. 1 μm and below) in a substrate for patch clamps and other purposes, the holes providing desirable shape and smoothness for creating gigaohm seals with cells.
At times it may be desirable to investigate temperature gradients around the patch clamp. This may be done by changing the temperature of the water bath in which the cells are held.